BZW2 promotes malignant progression in lung adenocarcinoma through enhancing the ubiquitination and degradation of GSK3β

The role of Basic leucine zipper and W2 domains 2 (BZW2) in the advancement of different types of tumors is noteworthy, but its involvement and molecular mechanisms in lung adenocarcinoma (LUAD) remain uncertain. Through this investigation, it was found that the upregulation of BZW2 was observed in LUAD tissues, which was associated with an unfavorable prognosis for individuals diagnosed with LUAD, as indicated by data from Gene Expression Omnibus and The Cancer Genome Atlas databases. Based on the clinicopathologic characteristics of LUAD patients from the tissue microarray, both univariate and multivariate analyses indicated that BZW2 functioned as an independent prognostic factor for LUAD. In terms of mechanism, BZW2 interacted with glycogen synthase kinase-3 beta (GSK3β) and enhanced the ubiquitination-mediated degradation of GSK3β through slowing down of the dissociation of the ubiquitin ligase complex, which consists of GSK3β and TNF receptor-associated factor 6. Moreover, BZW2 stimulated Wnt/β-catenin signaling pathway through GSK3β, thereby facilitating the advancement of LUAD. In conclusion, BZW2 was a significant promoter of LUAD. The research we conducted identified a promising diagnostic and therapeutic target for LUAD.

difluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA).Then we used 5% non-fat milk to block the membranes at room temperature for 1 h and subsequently incubated the PVDF membranes with primary antibodies at 4°C overnight after washed by TBST (thrice for 10 min each).The second day, a super chemiluminescence (ECL) kit (Yeasen, 36222ES, Shanghai, China) was used to visualize the target protein after the secondary antibody for 1 h at room temperature.
The antibodies are listed in Supplementary Table S3.

EdU incorporation assay
The cells that transfected for 24 hours were seeded into 96-well plates (8×10³ cells/well).After the cells were cultured to the normal growth stage, the medium in the 96-well plate was replaced with fresh medium containing 5-ethynyl-2-′deoxyuridine (EdU; 1000:1) for 2 h.The measurement was performed using EdU incorporation assay kit (RiboBio, C10310-1, Guangzhou, China) according to the manufacturer's protocol.
A fluorescence microscope (Nikon, Japan) was used to photograph the stained cells.

Flow cytometry analysis
A Cell Cycle Staining Kit (Multi Sciences, C10310-1, Hangzhou, China) was used to detect the cell cycle distribution of the cells that transfected for 24 hours through a flow cytometer according to the manufacturer's protocol.ModFit software was used to determine the percentage of cells in various phases of the cell cycle.

Wound healing assay
The cells that transfected for 24 hours were seeded into 6-well plates.When the confluence of these cells reaches to 90%, the tip of a 200-μL pipette was used to scratch the cell monolayer.After washed with PBS, the cells were cultured with serum-free media.The width of the wound was visualised under a light microscope at the indicated time.

Transwell assay
The upper Transwell chamber with or without Matrigel was seeded with the cells (3×10 4 cells/well) that transfected for 24 hours and the lower Transwell chamber was added with 700 µL of a medium supplemented with 20% FBS.After 24 h, the cells in the upper chamber moved to the lower chamber and were fixed with methanol for 30 min.Subsequently, the cells were stained with 0.2% crystal violet for 20 min and were photographed by a light microscope.

Lentivirus production and infection
The sh-NC, sh-BZW2, oe-NC and oe-BZW2 lentiviruses (Syngentech, Beijing, China) were transfected into the corresponding cells.The sh-RNA sequences are listed in Supplementary Table S1.

Data processing and transcriptome sequencing
Transcriptome sequencing (RNA-seq) was performed by Lianchuan Biotechnology using the Illumina HiSeq4000 platform.The samples were PC-9 cells transfected with siRNA including si-NC and si-1.We further performed computational simulation by using Ingenuity Pathway Analysis (IPA; QIAGEN, Valencia, CA, USA) online tools to canonical pathways.S2.Primers used for qRT-PCR.

Figure S2 .Figure S3 .
Figure S2.The expression of BZW2 in LUAD cell lines and bronchial epithelial cells.

Figure S5 .
Figure S5.BZW2 interacts with GSK3β.A The PPI network was obtained from the